title: Interactions of MCP1 with components of the replication machinery in mammalian cells.
abstract: Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia da Universidade do Porto, Portugal. elsa.rocha@ff.up.pt
authors.forename: Elsa
authors.initials: E
authors.lastname: Bronze-da-Rocha
authors.forename: Chii-Mei
authors.initials: CM
authors.lastname: Lin
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Shimura
authors.forename: Mirit I
authors.initials: MI
authors.lastname: Aladjem
mesh_terms: D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D018932:Chemokine CCL2 / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D002853:Chromatography, Liquid; D000090266:Chromobox Protein Homolog 5; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism; D011485:Protein Binding; D053719:Tandem Mass Spectrometry
chemical_list: C000715593:CBX1 protein, human; C111835:CCL2 protein, human; C113571:CDC45 protein, human; C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D018932:Chemokine CCL2; D004268:DNA-Binding Proteins; D006657:Histones; C066470:MCM3 protein, human; D009687:Nuclear Proteins; C497224:ORC2 protein, human; C109152:ORC4 protein, human; D051738:Origin Recognition Complex; D000090266:Chromobox Protein Homolog 5; C084859:MCM2 protein, human; C101624:MCM7 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064168:Minichromosome Maintenance Complex Component 7